Paromomycin antibiotic derivatives

ABSTRACT

Derivatives of paromomycin have been prepared which possess substantially improved antibacterial activity. An example of such an agent is 1-(L-(-)- gamma -amino- Alpha hydroxybutyryl)paromomycin (IVa,BB-K47).

United States Patent Naito et al.

[451 July 29,1975

PAROMOMYCIN ANTIBIOTIC DERIVATIVES Inventors: Takayuki Naito; SusumuNakagawa,

both of Tokyo, Japan Assignee: Bristol-Myers Company, New York,

Filed: June 15, 1973 Appl. No.: 370,280

Related [1.8. Application Data Continuationin-part of Ser. No. 250,389,May 4',

1972, abandoned.

US. Cl 260/210 AB; 424/180 Int. Cl. C07H 15/20 Field of Search 260/210AB [56] References Cited UNlTED STATES PATENTS 3,781,268 12/1973Kawaguchi ct a1 260/210 AB OTHER PUBLICATIONS Omoto The Journal ofAntibiotics, Vol. XXIV, N0. 7, 1971, pp. 430-434.

Primary Examiner-Johnnie R. Brown Attorney, Agent, or FirmRobert E.Havranek [57] ABSTRACT 6 Claims, No Drawings 1 PAROMOMYCIN ANTIBIOTICDERIVATIVES CROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of co pending application Ser. No. 250,389 filedMay 4, 1972 now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention This inventionrelates to semisynthetic l-substituted derivatives of paromomycin, saidcompounds being prepared by acylating the l-aminofunction of paromomycinwith y-amino-a-hydroxybutyryl, ,B-amino-a-hydroxypropionyl orfi-amino-ahydroxyvaleryl moieties.

2. Description of the Prior Art:

A. Paromomycin is an antibiotic described in U.S. Pat. No. 2,916,385,which issued Dec. 8, 1959. The patent describes the preparation andstructure of paromomycin and indicates the organism employed in thefermentation is deposited in the Culture Collection of the FermentationLaboratory, Northern Utilization Research and Development Division, U.S.Department of Agriculture, Peoria, Illinois as NRRL 2455.

B. The compound is further described in the Eighth Edition of MerckIndex on page 784.

SUMMARY OF THE INVENTION The compound having the formula CH On J fra aPR2 IV in which R is L-()-8-amino-a-hydroxybutyryl, L-(-)-,B-amino-oz-hydroxypropionyl or L-()-6-amino-ahydroxyvaleryl; or anontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceuticallyacceptable acid addition salt shall mean a mono, di-, tri, tetra orpentasalt formed by the interaction of one molecule of compound IV with15 moles of a nontoxic, pharmaceutically acceptable acid. Included amongthese acids are acetic, hydrochloric, sulfuric, maleic, phosphoric,nitric, hydrobromic, ascorbic, malic and citric acid, and those otheracids commonly used to make salts of amine containing pharmaceuticals.

Thecompounds of the present invention are prepared by the followingrepresentative diagramatic scheme:

N- (Benzyloxycarbonyloxy) l Paromomycin Succinimide I II -ContinuedN-Hydroxysuccinimide ester of L- 2.) Compound II benzyloxycarbonylaminowhydroxybutyric acid no CHQOH 3.) Compound IIIa H /Pd/C mic i c C Iva Apreferred embodiment of the present invention is 45 H; or a nontoxicpharmaceutically acceptable acid ad the compound having the formuladition salt thereof.

HO CH OH en-Ir OH C i h.11\ V in which R is H or 60 Another preferredembodiment is the compound 0 formula V in which R is O C.;H ,CH-,-Ol

5 C,H,-,CH2O-C and R is H, L-()-y-amino-a-hydroxybutyryl, L-()-B-amino-a-hydroxypropionyl or L-()-8-amino-ozand R is H. hydroxyvaleryl;wherein R and R must be other than A most preferred embodiment is thecompound c 5 formula V wherein R is H and R isL-()-y-amino-oehydroxybutyryl; or a nontoxic pharmaceutically acceptableacid addition salt thereof.

A most preferred embodiment is the compound of formula V wherein R is Hand R is L-(.)-,8-amino-ahydroxypropionyl; or a nontoxicpharmaceutically acceptable acid addition salt thereof.

A most preferred embodiment is the compound of formula V wherein R is Hand R is L-()-8-amino-othydroxyvaleryl; or a nontoxic pharmaceuticallyacceptable acid addition salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride,acetate, maleate, citrate, ascorbate, nitrate or phosphate salts ofcompound V.

Another most preferredembodiment is the monosulfate salt of compound V.

Still another most preferred embodiment is the disulfate salt ofcompound V.

The objectives of the present invention have been achieved, by theprovision according to the present in vention of the process for thepreparation of the compound having the formula in which R isL-(*)-y-amino-a-hydroxybutyryl, L- (-)B-amino-a-hydroxypropionyl orL-(-)-6-amino-0thydroxyvaleryl; or a nontoxic pharmaceuticallyacceptable acid addition salt thereof; which process comprises theconsecutive steps of A. treating paromomycin with an agent selected fromthe compounds having the formulas (or a carbodiimide addition compoundthereof) or (or a carbodiimide addition thereof), in which R and R are.alike or different and each is H, F, Cl, Br, N0 OH, (lower)alkyl or(lower)alkoxy, X is chloro, bromo or iodo, or a functional equivalent asa reactant; in a ratioof one mole or less of agent per mole ofparomomycin in a solvent, preferably selected from the group comprisedof dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane,1,2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine,N-(lower)al' kylpiperidine, or mixtures thereof, but preferably 1:1water-tetrahydrofuran, at a temperature below 50C. and preferably below25C., to produce the compound having the formula in which Y is a radicalof the formula CH 0 l R H 3 ll CH -O-C- CHCO\.

I o rz 2 O O Y I H n :5 or c-c'F -cn -cand X, R and R are as definedabove:

B. acylating compound II with an acylating agent having the formula OHOWNH(CH ),,H( M Vll Vll in which n is an integer of l to 3 inclusive andW is a radical selected from the group consisting of but preferably M isa radical selected from the group comprising O-N o 2 I a I 10 0 7preferably I in which R and R are as above; in a ratio of at least 0.5mole of compound Vll per mole of compound II, but preferably in a ratioof about 0.5 to about l.4, and most preferably in a ratio of about 0.8to about L1, in a solvent preferably selected from the group comprisinga mixture of water and ethyleneglycol dimethyl ether, dioxane,dimethylacetamide, dimethylformamide, tetrahydrofuran, propyleneglycoldimethyl ether, or the like but preferably 1 :1 watertetrahydrofuran, toproduce the compound of the formula \\LE, hO

It'll-(3 0 EEO-CH III in which n is an integer of l to 3, and Y and Ware as above; and

C. removing the blocking groups W and Y from compound III by methodscommonly known in the art, and preferably when W and Y are radicals ofthe formula by hydrogenating compound III with hydrogen in the presenceof a metal catalyst, preferably selected from the group comprisingpalladium, platinum, Raney nickel, rhodium, ruthenium and nickel, butpreferably palladium, and most preferably palladium on charcoal, in awater-water miscible solvent system, preferably selected from the groupcomprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethylether,

propyleneglycol dimethyl ether, or the like, but preferably 1:1water-tetrahydrofuran.

It should be apparent to those knowledgeable in the art that otheragents can be used in the process above essential. These reactions arewell-known in the art [cf. U.S. Pat. Nos. 3,079,314, 3,117,126 and3,129,224 and British Pat. Specification Nos. 932,644, 957,570 and959,054].

to acylate the amine functions ofthe intermediate com- C d Iv 1 [L-(-) ih d pounds of the instant invention. This disclosure isybutyryflparomomycin, possesses excellent antibactemeant to include allsuch acylating agents that produce rial activity. Illustrated below is atable showing the labile amine blocking groups, said labile blockingminimal inhibitory concentrations (MICs) of paromogroups commonlyemployed in the synthesis of pep mycin and compound lVa (BB-K47) againsta variety tides. The labile blocking groups must be readily reofgram-positive and gram-negative bacteria as 0bmovable by methodscommonly known in the art. EX- tained by the Steers agar-dilution method(Table l). amples of said labile blocking groups and their removalNutrient Agar Medium was used in the study of Table can be found in thereview of A. Kapoor, J. Pharm. Sci- 1. ences 59, pp. 127 (1970).Functional equivalents as In Vitro Antimicrobial Activities of BB-K47acylating agent for primary amine groups would inelude correspondingcarboxylic chlorides, bromides, MlC by Steers Method acid anhydrides,including mixed anhydrides and part g ticularly the mixed anhydridesprepared from stronger Bristol BB K47 Pammo aclds such as the loweraliphatic monoesters of car- Strain No. lot1-2 mycin bonic acid, ofalkyl and aryl sulfonic acids and of more E coli NIH] l 6 l 6 hinderedacids such as diphenylacetic acid. In addition, J hl A151 19 3 an acidazide or an active esterof thioester (e.g., with KM R* 25323 i1p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioace- .1 A9841 3.] ticacid) may be used or the free acid itself may be cou- KM-R* A20365 0.8100 pled with compound 11 after first reacting said free acid 11' M06641'2 with N,N'-dimethylchloroforminium chloride [cf. /141* 0 5 1,6 100 vd E W677 A20684 1.6 1.6 Great Britain 1,008,170 and No ak an Werchet, xBegum/W677 M0685 125 100 per1ent1a XXI/6,360 (1965)] or by the use ofenzymes NIH] O 8 L6 or of an N,N-carbonyldiimidazole or an N,N'-

P 3038 A20680 5 5: ype o. carbonylditrlazole [cf Sheehan and Hess, .I.Amer. Smarcescens Azoolg L6 L6 Chem. Soc., 77, 1067, (1955)] or ofalkynylamine rea- P. aeruginosa D 15 6.3 100 l gent [of R. Buljlle andH. G. Vrehe, Angew., Chem., H9 D 133 KM R A9923 Z188 InternationalEdition 3,582 1964)], or ofa ketenimine A9930 0.8 12.5 reagent [cf. C.L. Stevens and M. E. Monk, J. Amer. 1; acruglmsa D45 12132 Z188 Chem.Soc., 80, 4065 (1958)] or of an isoxazolium salt M0717 50 reagent [cf.R. B. Woodward, R. A. Olofson and H. M0718 50 P. vulgaris A9436 0.8 0.8Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. An- I! A9526 08 146 otherequivalent of the acid chloride is a corresponding 1". mirabilis 233(5):azolide, i.e., an amide of the corresponding acid with 40 R morgzmiiA9553 amide nitrogen is a member of a quasiaromatic five A20031 1.6 1.6membered ring containing at least two nitrogen atoms, aureus 53g; SM'RM,$2 i.e., imidazole, pyrazole, the triazoles, benzimidazole, KM-R* A202390.4 100 benzotrizole and their substituted derivatives. As an exlf g 1ample of the general method for the preparation of an azolide,N,N-carbonyldiimidazole is reacted with a 8; 8g carboxylic acid inequimolar proportions at room temperature in tetrahydrofuran,chloroform, dimethyl- KM*Kanamycin Resistant.

' GM**-Gentamicin Resistant. formamlde or a similar inert solvent toform the car sMmSmpwmycin Rcsismm boxylic acid imidazollde inpractically quantltative yield which liberation of carbon dioxide andone mole of imidazole. Dicarboxylic acids yield diimidazolides.Compounds IVb (BB-K 135) and IVc (BB-K 128) The by-product, imidazole,precipitates and may be were also assayed in vitro as compared tocompound separated and the imidazolide isolated, but this is not Wu andparomomycin as shown below:

MIC (meg/ml.)

Bristol Organism No. [Vb WC W2: Paromomycin E. coli NIHJ 1.6 1.6 0.8 1.6Juhl A1511) 3.1 1.6 0.8 1.6 A1516) 3.1 1.6 0.8 1.6 KM-R A20363 12.5 3.10.8 A9844 1.6 1.6 0.8 1.6 KM-R A20365 3.1 1.6 0.2 100 K-12 1.6 1.6 0.81.6 KM-R A20664 0.8 0.8 0.4 0.8 KM-R A20665 3.1 1.6 0.4 100 W677 A206841.6 1.6 0.4 1.6 JR/W677 A20683 12.5 2. 3.1 100 K. pneumoniae D-ll 0.40.8 0.1 0.4 Type 22 No. 3038 Azoeso 50 50 12.5 100 S. marccsccns A200191.6 1.6 0.8 1.6 P. acruginosa D15 0 6.3 6.3 50 H9 D-113 KM-R 100 100 100100 C ontinued MlC (mcg/ml.)

Bristol Organism No. lVb 1Vc 1Vu Paromomycin A9923 50 25 12.5 100 A99303.1 0.8 0.4 6.3 Al5l50 50 25 125 100 Al94 100 100 6.3 100 GM-R A20717100 100 12.5 100 GM-R A20718 100 50 125 100 P. vulgaris A9436 0.8 1.60.4 0.8 A9526 1.6 1.6 0.8 0.8 P. mirabilis A9554 3.1 1.6 1.6 1.6 A99003.1 0.8 1.6 1.6 P. morganii A9553 3.1 1.6 0.8 0.8 A2003] 1.6 1.6 0.8 1.6S. aureus Smith 04 0.2 0.2 0.2 2091 SM-R 1.6 1.6 0.8 1.6 KM-R A2023) 6.31.6 0.8 100 Mycobacterium 607 1.6 0.4 0.4 0.4 "KM-R 50 25 25 "KM,SM R 2512.5 12.5 12.5 phlei 0.4 0.2 0.1 0.2 ranae 1.6 0.4 0.4 0.4

Compound lVa generally appears more potent against the test organismsthan either lVb or IVc but all are still potent antimicrobial agents.

Compounds IV are valuable as an antibacterial agents, nutritionalsupplements in animal feeds, therapeutic agents in poultry and animals,including man, and are especially valuable in the treatment ofinfectious diseases caused by Gram-positive and Gramnegative bacteria.

Compounds 1V when administered orally are useful as an adjunctivetreatment for preoperative sterilization of the bowel. Both aerobic andanaerobic flora which are susceptible to these drugs are reduced in thelarge intestine. When accompanied by adequate mechanical cleansing, theyare useful in preparing for colonic surgery.

Compounds IV are effective in the treatment of systemic bacterialinfections when administered parenterally in the dosage range of about250 mg. to about 3,000 mg. per day in divided doses three or four timesa day. Generally the compounds are effective when administered at adosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Compounds of formula IV and the salts thereof are known to form monoandpolyhydrates upon isolation from aqueous solvents. Accordingly, thehydrates so produced are considered an integral part of the instantinvention.

EXAMPLES EXAMPLE 1 Preparation ofL-()-'y-Benzyloxycarbonylamino-a-hydroxybutyric Acid (Vla)L-()-y-amino-oz-hydroxybutyric acid (7.4 g., 0.062 mole) was added to asolution of 5.2 g. (0.13 mole) of sodium hydroxide in 50 m1. of water.To the stirred solution was added dropwise at 05C. over a period of 0.5hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride and the mixture wasstirred for 1 hour at the same temperature. The reaction mixture waswashed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloricacid and extracted with four -ml. portions of ether. The etherealextracts were combined, washed with a small amount of saturated sodiumchloride solution, dried with anhydrous sodium sulfate and filtered. Thefiltrate was evaporated in vacuo and the resulting residue wascrystallized from benzene to give 1 1.6 g. (74%) of colorless plates;melting point 78.579.5 C., [ab-4.5 (c 2, CH OH). Infrared (1R) lKBrl: c0 1740, 1690 cm. Nuclear Magnetic Resonance (NMR) (acetone-d )8 (in ppmfrom TMS) 2.0 (2H, m), 3.29 (21-1, d-d, J 6.7 and 12 Hz), 4.16 (1H, d-d,.1 4.5 and 8H2), 4.99 (2H,s), 6.2 (2H, broad), 7.21 (5H,s). The productis compound Vla.

Anal. calc'd for C H NO C, 56.91; H, 5.97; N,

5.53. Found: C, 56.66; H, 5.97; N, 5.47.

EXAMPLE 2 N-Hydroxysuccinimide Ester of L-()-y-benzyloxycarbonylamino-a-hydroxybutyric Acid (Vlla).

A solution of 10.6 g. (0.042 mole) of Vla and 4.8 g. (0.042 mole) ofN-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to 0 C. andthen 8.6 g. (0.042)mole of N,N'-dicyclohexylcarbodiimide was added. Themixture was kept overnight in a refrigerator. The dicyclohexylurea whichseparated was filtered off and the filtrate was concentrated to about 50ml. under reduced pressure to give colorless crystals of Vlla which werecollected by filtration; 6.4 g./m.p. l21122.5C. The filtrate wasevaporated to dryness in vacuo and the crystalline residue was washedwith 20 ml. ofa benzenem-hexane mixture to give an additional amount ofVlla. The total yield was 1.34 g. (92%). [01],, 1.5 (c 2, CHCl 1R(KBr) c0 1810,1755,

1740, 1680 cm. NMR (acetone-d )8 (in ppm from TMS) 2.0 (2H,m), 2.83(4H,s), 3.37 (211,d-d, J 6.5 and 12.5 Hz), 4.56 (1H,m), 4.99 211,5 6.3(2H,broad), 7.23 (5H,s).

Anal. calcd c,.,H,,,N o,-. C, 54.85; H, 5.18; N, 8.00. Found: C, 54.79,54.70; H, 5.21, 5.20; N, 8.14, 8.12.

1. G. W. Anderson et al.. J. Am. Chem. Soc, 86. 1839 (1964).

EXAMPLE 3 Preparation of 6"'-N-BenZyloxycarbonylparomomy cin (11) To astirred solution of 2.413 g. (3.92 m. mole) of paromomycin free base in55 ml. of 45% aqueous THF (tetrahydrofuran) was added 975 mg, (3.92 m.mole) of N-benzyloxycarbonyloxysuccinimide at 10 C. The reaction mixturewas stirred overnight at room temperature and evaporated under reducedpressure to remove the organic solvent. The resultant aqueous solutionwas filtered to remove any insoluble material and the filtrate wascharged on a column of Amberlite CG-SO resin (NH 60 ml. [Amberlite]. Thecolumn was washed with 350 ml. H and eluted with 0.1 N Nl-1 OH. Theeluate was collected in 20 mL-fraction. Fractions 13 to 21 werecombined, evaporated under reduced pressure and lyophilized to give 1.19g. (40%) of crude 6"-benzyloxycarbonylparomomycin (11) which did notcontain paromomycin itself. 1700 cm. The product was used for the nextreaction with out further purification.

EXAMPLE 4 Preparation of1-[L-(-)-y-benzyloxycarbonylaminoa-hydroxybutyryl]-6"-carbobenzoxyparomomycin(Illa) To a stirred solution of 1.16 g. 1.55 m. mole) of 6"-benzyloxycarbonylparomomycin in 30 ml. of 50% THF and water was added542 mg. (1.55 m. mole) of N-hydroxysuccinimide ester ofL-(-)-ybenzyloxycarbonylamino-a-hydroxybutyric acid at 10 C. Thereaction mixture was stirred for 5 hours at room temperature to yield amixture of N[L-(-)-ybenzyloxycarbonylamino-oz-hydroxybutyryl]-6"carbobenzoxyparomomycins of whichl-[L-()-'ybenzyloxycarbonylamino-oz-hydroxybutyryl]-6"'-carbobenzoxyparomomycin (11111) was a major component.

EXAMPLE 5 Preparation of 1-[L-()-y-amino-a-hydroxybutyryl] paromomycin(lVa) The crude product 111a from example 4 was hydrogenated underatmospheric pressure in the presence of 200 mg. of 10% palladium oncharcoal overnight at room temperature. The reaction mixture wasfiltered and evaporated under reduced pressure to remove the organicsolvent. The resultant aqueous solution was adsorbed on a column ofAmberlite CG-SO (NH.,*, 37 ml.). The column was washed with water andirrigated successively with 600 ml. of 0.1 N ammonia, 200 ml. of 0.2 Nammonia, 300 ml. of 0.3 N ammonia, 700 ml. of 0.5 N ammonia and finally700 ml. of l N ammonia. The eluate was collected in 10-ml. fraction. Thefractions were divided into the following groups by ninhydrin test,bioassay (B. subtill's) and TLC (silica gel, [S-

114 C1-1Cl -C1-l O1-l28%NH OH-H O 1:4:2:1, ninhydrin). The fractionsbelonging to the same group were combined, concentrated under reducedpressure and lyophilized.

Fraction Group No. Eluted by Wt. isolated Compound 1 70-77 0.2N NH ,OH30 mg. BB-K49 (crude)* 2 93-101 0.3N NH ,Ol-1 264 Paromomycin 3 109-1160.3N NH OH l7 BB-KSO 4 129-133 U.SN NH OH BB-K47 (lVa) 5 139-142 0.5N NH,OH 29 BB-KSl 6 197-202 1N NH OH 91 BB-K48 *Rechromatography withAmberlite CG-50 (NHf. 8 m1.) gave 17 mg. of pure BB-K49.

Rechromatography with Amberlite CG-SO (NHI. 10 ml.) gave 97 mg. ofa pureproduct designated Eli-K47, lot 1-2, the desired title product. 1[L-(-)-y-amino-a-hydroxybutyryl)paromomycin (lVa).

Properties:

Code No. M.p.(dec.) RF yC=O(KBr) [a]D(H O) BB-K47 (lVa) -188C. 0.171640""" +58 (lot 1-2) BB-K 48 -192 0.07 1640 BB-K 49 260 0.27 1690 1640BB-KSO 173-178 0.27 1640 BB-K 51 185-189 0.18 1640 TLC: silica gelplate. CHC1 -MeOH-28%NH OH-H O (1:4:211). Inexplicable for two carbonylbands which did not change by repeating hydrogenation.

BB-K47 anal. calcd. for C H N O H CQ 1/2H O:C,42.69; H, 7.04; N, 10.69.

Found: C, 42.69;

1t has been confirmed by TLC that all of the products, BB-K47 to BB-KS lgave paromomycin and whydroxy-y-aminobutyric acid by heating for 1.5hours in 0.5 N sodium hydroxide solution.

EXAMPLE 6 Preparation of N-(Benzyloxycarbonyloxy)succinimideN-hydroxysuccinimide (23 g., 0.2 mole) was dissolved in a solution of9g. (0.22 mole) of sodium hydroxide in 200 ml. of water. To the stirredsolution was added dropwise 34 g. (0.2 mole) of carbobenzoxychloridewith water-cooling and then the mixture was stirred at room temperatureovernight to separate the carbobenzoxy derivative which was collected byfiltration, washed with water and air dried. Yield 41.1 g. (82%).Recrystallization from benzene-n-hexane (10:1) gave colorless prismsmelting at 78-79 C.

EXAMPLE 7 Preparation of L-( )-'y-amino-a-hydroxybutyric acid fromambutyrosin A or B or mixtures thereof EXAMPLE 8 Preparation ofL-()-y-amino-a-hydroxybutyric acid fromDL-a-hydroxy-y-phthalimidobutyric acid.

A. Dehydroabiethylammonium La-hydroxy-'yphthalimidobutyrate: To asolution of 25 g. (0.1 mole) of a-hydroxy-y-phthalimidobutyric acid in200 ml. of ethanol was added a solution of 29 g. (0.1 mole) ofdehydroabiethylamine in 130 ml. of ethanol. The solution was shakenvigorously for a minute and stood at room temperature for hours duringwhich time fine needles crystallized out. The crystals were collected byfiltration, washed with 50 ml. of ethanol and airdried to obtain 30.1 g.(56%) of a diastereomer of the dehydroabiethylamine salt. M.p. 9394 C.[01],, (C. 2.5, MeOH). Recrystallization from 300 ml. of ethanol gave23.2 g. (43%) of the pure product. M.p. 94-95 C. [a] +lO.8 (C. 2.5MeOH). Further recrystallization did not change the melting point andthe specific rotation.

Anal. calcd. for C H N O .H O: C, 69.54; H, 8.02;

N, 5.07. Found: C, 69.58; H, 8.08; N, 5.07. 1. Y. Saito et al.,Tetrahedron Letters, 1970, 4863.

B. L-()-'y-amino-a-hydroxybutyric acid: To a solution of 1.5 g. (0.014mole) of sodium carbonate in 40 ml. of water were added 5.3 g. (0.01mole) of dehydroabietylammonium La-hydroxy-'yphthalimidobutyrate and 60ml. of ether. The mixture was shaken vigorously until all of the solidhad dissolved. The ether layer was separated. The aqueous solution waswashed twice with -ml. portions of ether and evaporated to 15 ml. underreduced pressure. To the concentrate was added 10 ml. of concentratedhydrochloric acid and the mixture was refluxed for 10 hours. Aftercooling, separated phthalic acid was removed by filtration. The filtratewas evaporated under reduced pressure. The residue was dissolved in 10ml. of water and filtered to remove a small amount of insoluble phthalicacid. The filtrate was adsorbed on a column of Amberlite lR-l20 (NHJ, 1cm. X 35 cm.), the column was washed with 300 ml. of water and elutedwith l N ammonium hydroxide solution. The eluate was collected in l5-ml.fraction. The ninhydrin positive fractions 10 to 16 were combined andevaporated under reduced pressure to give a syrup which recrystallizedgradually. The crystals were triturated with ethanol, filtered and driedin a vacuum desiccator to give 0.78 g. (66%) ofL-()-y-amino-a-hydroxybutyric acid. M.p. 206207 C. [01],, 29 (C. 2.5, H0). The IR spectrum was identical with an authentic sample which wasobtained from ambutyrosin.

EXAMPLE 9 Preparation of the Monosulfate Salt of l-[L()-y-amino-a-hydroxybutyryl]paromomycin.

One mole of l-[L-()-y-amino-aamino-a paromomycin is dissolved in 1 to 3liters of water. The solution is filtered to remove any undissolvedsolids. To the chilled and stirred solution is added one mole ofsulfuric acid dissolved in 500 ml. of water. The mixture is allowed tostir for 30 minutes, following which cold ethanol is added to themixture until precipitation occurs. The solids are collected byfiltration and are determined to be the desired monosulfate salt.

EXAMPLE 10 Preparation of the Disulfate Salt ofl[L-()-y-amino-a-hydroxybutyryllparomomycin One mole ofl-[L-(-)-'y-amino-a-hydroxybutyryl]- paromomycin is dissolved in 1 to 3liters of water. The solution is filtered to remove any undissolvedsolids. To the chilled and stirred solution is added 2 moles of sulfuricacid dissolved in I00 ml. of water. The mixture is allowed to stir for30 minutes. following which cold ethanol is added to the mixture untilprecipitation occurs. The solids are collected by filtration and aredetermined to be the desired disulfate salt.

EXAMPLE 11 Preparation of L-B-Benzyloxycarbonylamino-ahydroxypropionicacid (Vlb) L-B-amino-a-hydroxypropionic acid* (8.2 g., 0.078 mole) wasdissolved in a solution of 6.56 g. (0.0164 mole) of sodium hydroxide inml. of water. To the stirred solution was added dropwise 14.7 g. (0.086mole) of carbobenzoxy chloride below 5 C. The mixture was stirred for anhour at room temperature, washed with 60 ml. of ether and adjusted to pH2 with dilute HCl. The precipitate was collected by filtration, washedwith water and air-dried to give 9.65 g. (52%) of Vlb. The filtrate wasextracted with five l00-ml. portions of ether. The ethereal solution waswashed with water, dried over sodium sulfate and evaporated to drynessin vacuo to give additional 2.0 g. (l 1%) of Vlb. A total of 11.65 g. ofVlb was crystallized from 500 ml. of benzene-ethyl acetate (4:1) to give9.36 g. (50%) of pure Vlb; m.p. l28.5-l29.5 C. Infrared (IR) (KBr): 'y1745, 1690 cm". [01 +2.9 (c 5.0, MeOH). Nuclear Magnetic ResonanceSpectra [NMR (DMSO- d 6 (in ppm) 3.05-3.45 (2H, m, CH N), 4.05 (1H, d-d,O-CHCO), 5.03 (2H, s, CH Ar) 7.18 (1H, broad, NH), 7.36 (5H, 5, ring H).

Anal. calcdd for C H NO z C, 55.23; H, 5.48; N,

5.86. Found: C, 55.34; H, 5.49; N, 5.87. *K. Freudenberg. Ben. 47, 2027(1914).

EXAMPLE 12 Preparation of N-Hydroxysuccinimide Ester of L-B-benzyloxycarbonylamino-a-hydroxypropionic acid (Vllb).

To a chilled and stirred solution of 478 mg. (2 m.moles) of Vlb and 230mg. (2 m.moles) of N- hydroxysuccinimide in 10 ml. of tetrahydrofuran(THF) was added 412 mg. (2 m.moles) of dicyclohexylcarbodiimide. Themixture was stirred for an hour at 05 C., for 2 hours at roomtemperature and then filtered to remove the N,N-dicyclohexylurea. Thefiltrate containing Vllb was used for the next reaction withoutisolation.

EXAMPLE 13 Preparation of 1-[L-()-B-amino-a-hydroxypropionyl]paromomycin (lVb, BIZ-K).

To a stirred solution of 750 mg (1 m mole) of6"'-N-benzyloxycarbonylparomomycin (11) in 10 ml of water was added dropwise asolution of the activated ester Vllb in 10 m1 of THF which was preparedfrom 239 mg (1 m mole) of B-benzyloxycarbonylamino-oz-hydroxypropionicacid. The reaction mixture was stirred overnight and then hydrogenatedovernight with 200 mg of 10% palladium on charcoal at room temperatureat at mospheric pressure. The catalyst was filtered off and washed withwater. The filtrate and washings were combined and concentrated in vacuoto remove most of the organic solvent. The aqueous solution was adjustedto pH 7 with 1 N hydrochloric acid and passed through a column ofAmberlite 0050 (NH,*, 15 ml), which was washed with 500 ml of water andthen eluted with 700 ml of 0.1 N NH OH and 1 1 of0.2 N NH OH. The eluatewas collected in 10 -ml fraction. Tube Nos. 97l06 which showed Rf 0.23by TLC with S-l10 systern (ninhydrin) and activity against Pseudomonasaeruginosa were combined, evaporated in vacuo and freeze-dried to give34.7 mg (4.7%) of Nb; m.p. 181-186C (dec). IR (KBr): 1650, 1560 cm".

Anal. calcdd for C H N O .2H CO C, 40.68; H, 6.58; N, 10.16. Found: C,40.69; H, 6.44; N, 10.25.

EXAMPLE 14 Preparation of L-S-Benzyloxycarbonylamino-z hydroxyvalericacid (Vlc) To a stirred solution of 400 mg (3.0 m moles) ofL-8-amino-a-hydroxyvaleric acid* and 250 mg (6.5 m moles) of sodiumhydroxide in 25 m1 of water was added dropwise 580 mg (3.3 m moles) ofcarbobenzoxy chloride over a period of 30 minutes at 05C. The mixturewas stirred for an hour at -15C, washed with 25 ml of ether, adjusted topH 2 with hydrochloric acid and extracted with three 30-ml portions ofether. The combined ethereal solution was shaken with 10 ml of asaturated sodium chloride solution, dried over anhydrous sodium sulfateand evaporated in vacuo to give moles) of Vlc and 230 mg (2.0 m moles)of N- hydroxysuccinimide in ml of ethyl acetate was added 4 l 2 mg (2.0m moles) of N, N'-dicyclohexylcarbodiimide (DCC). The mixture wasstirred for 3 hours at room temperature and filtered to removeprecipitated N, N'-dicyclohexylurea. The filtrate was evaporated invacuo to yield 780 mg of viscous syrup (Vllc). lR(Neat): 'y 1810, 1785,1725 cm.

EXAMPLE 16 2 Preparation of 1[L-()-5-amino-a-hydroxyvaleryl] paromomycin(IVc, [BB-K128).

To a stirred solution of 750 mg (1 m mole) of 6-N-benzyloxycarbonylparomomycin (11) in 10 ml of water was added dropwise asolution of the activated ester Vllc in 10 ml of THF which was preparedfrom 267 mg (1 m mole) of L-6-benzyloxycarbonylamino-ahydroxyvalericacid. The mixture was stirred overnight at room temperature and thehydrogenated for 4 hours with 200 mg of palladium charcoal at roomtemperature at ordinary pressure. The catalyst was removed by filtrationand washed with water. The filtrate and washings were evaporated invacuoto remove most of the organic solvent. The resultant aqueoussolution was adjusted to pH 7 with 1 N hydrochloric acid and adsorbed ona column of Amberlite CG-SO (NH 15 ml), which was washed with 200 ml ofwater and then eluted with each 1,000 m1 of 0.1 N NHqOH, 800 ml of 0.2 NNH OH, 500 m1 of 0.5 N NH OH and 500 ml of 1.0 N NH OH. The eluate wascollected in lO-ml fraction and divided into the following appropriatefractions on the basis of Rf value by TLC (S-1 10, ninhydrin) and diskassay (Bacillus subtilis and Psuedomonas aeruginosa). Each fraction wasevaporated in vacuo and the residue was freeze-dried.

Fraction Tube No. NH OH Weight BB-K No. Identity 1 1 l7130 0.2 N 101.5mg paromomycin l 2 198-204 0.5 N 34.3 mg lVc l-N-acylated compound (4.770) 3 244-255 1.0 N 33.8 mg BB-K 129 diacylated compound (4 7(Physico-chemical data:

BB-K No. IR (KBr) Mp Rf value (S-l l0, ninhydrin) BB K 128 1640, 1570 cm179185C 0.21 BB'K 129 1640, 1560 cm 184188C 0.09

Found: C, 40.73;

crystals which were recrystallized from benzene to yield 631 mg (78%) ofVlc; m.p. l 1 1C; infrared spectrum [lR(l(Br)]: 3460, 3350, 1725, 1685,1535, 1280, 730, 690cm. Nuclear magnetic resonance spectrum[Nl\/1R(acetone-d,,)] 8 (in ppm) l.70(4H, m) 4.14(2H, q, J=4.5Hz),4.19(1H, m), 4.82(2H, s), 6.2(3H, broad), 7.25(51-l, s). [01],, 1.6 (c10, MeOH).

Anal. Calcd for C,,H,,N0,; C, 58.42; H. 6.41; N,

5.24. Found: C, 58.36; H, 6.50; N, 5.27.

*S. Ohshird et al., Yakugaku Zasshi, 87, 1 184 (1967).

EXAMPLE 15 N-Hydroxysuccinimide ester of L-S-benzyloxycarbonylamino-a-hydroxyvaleric acid (Vllc).

To a stirred and chilled solution of 535 mg (2.0 m

We claim: 1. A compound having the formula 1 CH NHR in which R is H orand R is H, L-()-B-amino-a-hydroxypropionyl, L-

benzyloxycarbonylamino-a-hydroxypropionyl, or L-()-5-benzyloxycarbonylamino-a-hydroxyvaleryl,

wherein R or R must be other than H; or a nontoxic pharmaceuticallyacceptable acid addition salt thereof.

2. The compound of claim 1 wherein R is MFR? and R is H.

3. A compound of claim 1 wherein R is C H CH OC and R is L-()-B-benzyloxycarbonylamino-a-hydroxypropionyl or L-()-5-benzyloxycarbonylamino-oc-hydroxyvaleryl.

4. A compound of claim 1 wherein R is H and R isL-(-)-fi-amino-a-hydroxypropionyl or L-()-6-aminoa-hydroxyvaleryl; or anontoxic pharmaceutically acid addition salt thereof.

5. The compound of claim 4 wherein R is H and R isL-()-B-amino-a-hydroxypropionyl; or the monoor disulfate salt thereof.

6. The compound of claim 4 wherein R is H and R isL()-5-amino-a-hydroxyvaleryl; or the monodisulfate salt thereof.

l k l Patent No Dnted July 22 975 lnvpntofls) Takayukl Nalto, SusumuNakagawa and SOlChlIO Toda It is corLiI'iod Llmlv (*rror appears in tho:1hoveil0ntifi0d patent and that said LoLLvrs Patvnt nru herebycorrected as shown below:

. On the Cover Shoot, in item [75] the following should be addod:

Soichiro Toda, Kasukabeshi Saitama-ken, Japan Signed and Se-aled'thiseleventh Of November I 975 ISEALI RUTH MASON MARSHALL [)ANN

1. A COMPOUND HAVING THE FORMULA
 2. The compound of claim 1 wherein R1is
 3. A compound of claim 1 wherein R1 is C6H5CH2-O-C- and R2 is L-(-)-Beta -benzyloxycarbonylamino- Alpha -hydroxypropionyl or L-(-)- delta-benzyloxycarbonylamino- Alpha -hydroxyvaleryl.
 4. A compound of claim 1wherein R1 is H and R2 is L-(-)- Beta -amino- Alpha -hyDroxypropionyl orL-(-)- delta -amino- Alpha -hydroxyvaleryl; or a nontoxicpharmaceutically acid addition salt thereof.
 5. The compound of claim 4wherein R1 is H and R2 is L-(-)- Beta -amino- Alpha -hydroxypropionyl;or the mono- or disulfate salt thereof.
 6. The compound of claim 4wherein R1 is H and R2 is L-(-)-delta -amino- Alpha -hydroxyvaleryl; orthe monodisulfate salt thereof.